The present invention relates to a method for direct detection of analytes using observable spectral changes in monomolecular films which occur upon the analytes selective binding to the film.
Analytical Chemistry
Analytical chemistry techniques have been used for many years to determine such medical parameters as hematocrit levels. While useful in their own right, analytical chemistry methods are of limited or no practical applicability to many biological parameters in which assessment would be valuable. Unless expensive and cumbersome gas chromatography methods are used, large quantities of analytes are generally required to accomplish such methods. Often, quantitative results are limited or not available. However, such techniques have been used for such basic chemical tests as creatinine assays.
Microbiological and Pathology Methods
Another approach to medical-biological systems analysis has been direct microscopic observation using various cell-staining and classic pathology techniques. Augmenting these capabilities have been well developed microbiological techniques, such as culturing, colony characterization, and observation of metabolic and nutrient limitations. Most of medical science has been developed using this basic arsenal of analytic techniques.
While culturing and direct tissue observation techniques have served as the bulwark of medical detection processes for many years, they have considerable limitations. Pathological analysis of patient tissues to determine the development of a disease state and the identification of the causative pathogen generally requires an invasive procedure. On the other hand, culturing the pathogen from various body fluid or other samples is time consuming and expensive.
Immunoassays
A breakthrough in medicine occurred with the development of immunoassay techniques. In these methods, an antibody is developed which will specifically bind to a target of interest. While costly in both their development and production, antibodies from animals allowed a very accurate analysis of a number of analytes which had previously been virtually unassessable in both research and particularly clinical situations.
An important technical advancement in immunoassay was the development of monoclonal antibodies. Instead of subjecting an animal to an analyte and harvesting its whole range of antibodies, in this techniques a single spleen cell of a sensitized animal is rendered immortal and multiplied many times. The resulting cell line is then cultured to produce a very specific and pure antibody product.
Because the antibody itself is a small molecule, it must be labeled in some way so that the binding event can be detected. This can be done with a dye, flourescent, radioactive or other label. Conversely, if binding inhibition occurs between a known amount of introduced, labeled analyte and the material to be analyzed, the diminution of the signal will indicate the presence of test analyte. If the agglutination of the antibody particles is of sufficient volume and density, the formation of a precipitant can also serve to signal the presence of an analyte.
In recent years, the research and medical communities have come to rely heavily on immunoassay techniques to detect and quantify biological materials. While successful in many respects, the indirect nature of immunoassay methods as well as their dependence on antibody materials, results in a variety of complications, problems, and assay limitations. Briefly, the development and production of antibodies remains expensive, and these molecules are sensitive to environmental changes. Also, only those materials to which antibodies can be produce can be detected by these systems.
Immobilization of Assay Components
Many types of analytical chemistry techniques can be optimized and their implementation expedited by the immobilization of one or more of the components of a reaction. For instance, if the material to be tested is present in only a small quantity in a test sample, the analyte may be at so small a concentration that it is beyond the detection capabilities of a particular assay system.
Many immobilizing materials are available. These materials have been used extensively in analytical chemistry procedures for such purposes as the concentration materials. Sephadex columns are very commonly used for such purposes. Except for their specific binding properties, it is preferred that immobilization materials are relatively inert so that they themselves do not interact in the test reaction or otherwise interfere with the assay. Another important quality for immobilization materials is that they be regular in their structure, so as to provide predictability in the testing situation.
Classically, immobilization has been accomplished on columns, liposomes or other surfaces. The use of such materials provides many advantages for an assay system. For instance, these materials allow easy segregation of reactants from the surrounding test washes. Bi-lipid layer surfaces on support structures, as is described below, are also serving an immobilizing role in analytical systems.
In a typical immobilization scheme, the analyte is concentrated by adhesion to a column for which it has affinity. The testing can then take place on a limited area surface, rather than in the defused three-dimensional array of the original sample fluid. The reaction of the results are then concentrated in a smaller area, and are more likely to reach the level of detectability. A number of iterations of this technique are equally applicable to assay systems. These include concentrating the bound reactants to achieve an intensified signal, binding the signal producer and reacting the analyte on that surface, etc.
Immobilization techniques have proven very useful in the case of immunoassay approaches. Some of the difficulties with immunoassays lie in the submicroscopic nature of these materials. In a free-floating system, it is difficult to separate various parts of a sample which can obscure results, and to assure the maintenance of critical materials. For instance, there may only be a small amount of analyte in a sample, and the antibody can be very expensive, so that pure agglutination procedures will not accomplish a practical assay.
In response to these limitations, the research community has developed means of "immobilizing" the various components of an assay, both to concentrate analyte, and to localize a binding event. Basically, one component of the test is "tacked down" to a surface, and anything which subsequently binds to that component is likewise immobilized. This approach allows many advantages in using immunoassay techniques to their full potential.
An example of the use of immobilization in immunoassay techniques is where a test sample is washed across a surface to which it binds. An antibody is then washed across this treated surface, allowing specific immunogenic binding to occur. The antibody may have been pre-treated with a tag, in which case a color change, fluorescences or other such label is observed in a small, limited area. This approach provides maximum efficiency by limiting the amounts of both analyte and test materials required.
Bilayer Films as Immobilizing Supports
Bilayer films on surfaces have been used to provide the qualities of relatively expensive film materials to low cost support bases. Chemical modification of surfaces by organic monomolecular films has recently been used in an effort to develop such new materials. The ultrathin film coatings which can be achieved by these new approaches can effectively alter the surface properties of the original underlying material.
Because of these motivations, the techniques of molecular self-assembly, such as that described by Swalen et al., (Langmuir, Vol. 3, page 932, 1987) as well as Langmuir-Blodgett (LB) deposition (Roberts, Ed. Langmuir-Blodgett Films, Wiley, New York, 1966) are being used for coating surfaces with a well-defined, quasi two-dimensional array of molecules. The initial use for this new advancement was for materials science applications such as wetting (Whitesides, et al., Langmuir, Vol. 6, p. 87, 1990) and friction (Novotny et al., Langmuir Vol. 5, p. 485, 1989).
These bilayer films are also being used as immobilizing supports for analytic reactions. Bio-sensors based on LB films can detect molecules of diagnostic significance such as glucose (Okahata, et al., Thin Solid Films, Vol. 180, p. 65, 1989) and urea (Arisawa, et al., Thin Solid Films, Vol. 210, p. 443, 1992). In these cases, classic analytical chemistry systems are immobilized on the films in order to improve the readout of the test results and otherwise simplify and improve the detection capabilities of the test procedure.
The detection of receptor-ligand interaction is generally accomplished by indirect assays such as the enzyme-linked immunosorbent assay. Although biotechnological functionalized films have led to elegant examples of molecular recognition at an interface, the problem of transducing the molecule recognition event into a measurable signal has remained a difficulty until the advent of the subject invention.
In the case of biosensor devices, detection is generally carried out by coupling the LB films to a secondary device such as an optical fiber (Beswick, Journal Colloid Interface Science, Vol. 124, p. 146, 1988), quartz oscillator (Furuki et al., Thin Solid Films, Vol. 210, p. 471, 1992), or electrode surfaces (Miyasaka, et al., Chemical Letters, p.627, 1990).
Some of the analytes bound to these films provide for fluorescent label, where the fluorescence or its quenched state indicate the occurrence of a binding event (Beswick, Journal Colloid Interface Science, Vol. 124, p. 146, 1988). In some cases, these detection materials have been embedded in the surface of the supporting bi-lipid layer (Tieke, Advanced Materials, Vol. 3, p. 532, 1991).
Polydiacetylene films are known to change color from blue to red with an increase in temperature or changes in pH due to conformational changes in the conjugated backbone (Mino, et al., Langmuir, Vol. 8, p. 594, 1992; Chance, et al., Journal of Chemistry and Physics, Vol. 71, p. 206, 1979; Shibutag, Thin Solid Films, Vol. 179, p. 433, 1989; Kaneko, et al., Thin Solid Films, Vol. 210, p. 548, 1992). While it has been a goal of the research community to exploit this characteristic in the detection of binding events, researchers have yet to develop a method using this phenomenon in practical applications.
It would be highly desirable if the direct detection method of analytical chemistry techniques could be achieved with very small and biological molecules present in minute amounts in the analytic fluid, as this would represent a revolution in the bio-medical analytic arts. It would be ideal if the technology of monomolecular film supports could be developed in a unique way so that the binding event causes a change in the support material that could be directly detected.